1. Field of Invention
The present invention provides pharmaceutical compositions and methods of use for therapy of neurological inflammatory diseases with (5′-deoxy-5′-adenosyl)cobamamide, by itself or in combination with recombinant human growth hormone, Interleukins IL-1, IL-6, I′L-11, Epidermal Growth Factor, and physiotherapy.
2. Prior Art
The current generation of therapeutic agents and treatments for multiple sclerosis (MS) and other related neurological diseases seeks to reduce incidence and severity of lesions and to minimize clinical relapses. The current therapeutic methods have little effect on regeneration and remyelination. The etiology of demyelinating diseases is not well understood. Virtually all therapeutic agents were initially developed utilizing the Experimental Allergic Encephalomyelitis (EAE) animal model. The EAE animal model is an extreme example of central nervous system inflammation. When an etiological hypothesis is proposed, a therapy can be designed and implemented to address that proposed etiology. If the etiological hypothesis is essentially correct, then the implemented therapy will be effective. Without being restricted to any specific etiology, an original etiological hypothesis for MS is proposed and this provides a basis for therapeutic design. Specific therapeutic methods are described to address this etiological hypothesis. A scoping experiment in therapy, addressing a central feature of this hypothetical etiology, has been accomplished with a clinically definite MS patient who is in the Secondary Progressive stage of MS (SPMS). The dramatic improvement in the patient's condition strongly supports the etiological hypothesis. The recovery of physical abilities lost years before may indicate that remyelination occurred. It is doubtful that the EAE animal model could well demonstrate the effectiveness of this therapy.
The Etiology of Multiple Sclerosis
Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system (CNS). There is general agreement that MS is an autoimmune disease in which B cells, T cells, and macrophage immune system cells enter the CNS, after a breach of the blood brain barrier (BBB), and begin the inflammatory and demyelinating pathological process. There have been thousands of publications describing research on MS. However, none has convincingly identified the exogenous antigenic environmental factors that initiate the cascade of immune system events that ultimately becomes the MS disease state. Thus, the etiology of MS has remained unknown.
The accumulated research logically demands that there is a particular exogenous antigen, or a class of exogenous antigens, bearing responsibility for the fundamental and seminal CNS immune response that leads to the development of MS. This patent describes an etiological hypothesis, strongly supported by published research, which allows design of therapeutic methods for the control of and recovery from MS.
The hypothetical etiology of multiple sclerosis begins with an infection in the systemic circulatory system by microbes, wherein said microbes contain enzymes for the production of (5′-deoxy-5′-adenosyl) cobamide, which is referred to as 5′-deoxyadenosylcobalmin (AdoCbl). Some of the microbial enzymes that produce AdoCbl might be classified as adenosyltransferase enzymes. Said microbial enzymes are highly homologous to human ATP:Cob(I)alamin adenosyltransferase (referred to as adenosyltransferase or as ATR). Many bacteria produce and utilize AdoCbl, and therefore have numerous enzymes that bind AdoCbl, as does human ATR. When systemic B lymphocytes bind to a microbe, an Antigen Presenting Complex (APC) is formed after which a T-helper cell activates the B lymphocyte making it replication competent, and resulting in clonal expansion. Eventually, the microbe is destroyed, its cell wall and cytoplasmic membrane are ruptured and then enzymes from the interior of the microbe are released. The various enzymes are also bound by B lymphocytes thereby producing APCs, and ultimately resulting in clonal expansion of these B lymphocytes. An activated B lymphocyte produces immunoglobulins (Ig) commonly called antibodies. Placental mammals produce five different isotypes called IgA, IgD, IgE, IgG, and IgM. Antibodies to bacterial enzymes and/or viral antigens that are similar to human ATR are cross-reactive to human ATR. Thus, MS is an autoimmune disease, wherein the immune system attacks some part of the body, referred to as a self-antigen.
One fact demanding an exogenous antigen is the epidemiological hotspot in Olmstead County, Minnesota, with 177 cases of MS per 100,000 people, which is much higher than the expected 30 to 80 cases of MS per 100,000 people. Soybean production is the major agricultural business in Olmstead County. Soybeans are legumes. Legumes have root nodules with nitrogen fixing bacteria in a symbiotic relationship with the legume plant, especially bacteria in the genus Rhizobium. The Rhizobia bacteria produce and utilize AdoCbl. The bacterial enzymes are somewhat homologous to human ATR. Therefore, antibodies to the bacterial ATR-like enzymes will be cross-reactive to human ATR. A soil sample contains a large number of different bacteria. A person with a cut, which has been contaminated with soil, will thus have many B lymphocytes activated against the bacteria, and subsequent to the destruction of the bacteria, against the enzymes released from the interior of the bacteria.
There are many bacteria that have ATR-like enzymes and each enzyme might have antibodies produced to it, which are cross-reactive to human ATR. An example with IgG antibodies could be described as % homology to human ATR equals approximate % cross-reactive antibody to human ATR, where a fraction is used for each antibody's reactivity:
      IgG    ⁢          -        ⁢    Bacillus    ⁢                  ⁢    subtilus        IgG    ⁢          -        ⁢    human    ⁢                  ⁢    ATR  In this example, Bacillus subtilus is 41% homologous to ATR and the fractional representation in decimal form would be 0.41. Actual cross-reactivity may not be 41% as it depends on the active regions, but this is for discussion. Suppose there are four (4) ATR-like enzyme infections for which the immune system is on surveillance, IgG1=0.41, IgG2=0.32, IgG3=0.37 and IgG4=0.39. Thus, the cross-reactivity to ATR would beIgGATR=Σ0.41+0.32+0.37+0.39=1.49
Any one bacterial IgG might not strongly react to ATR; however, the collection of IgGs is highly reactive to ATR. This cumulative effect could be a factor in the difficulty finding an exogenous antigen responsible for the development of MS. The ATR-like enzymes are a class of bacterial enzymes; therefore, there is not a single specific microbe, common to all MS patients. One other point may have obscured identification of an anti-ATR immune response. In the body, as a whole, the ATR enzyme is located predominantly in the interior of mitochondria. Therefore, the inner and outer membranes of the mitochondrion protect the ATR enzyme from an immune attack. Genetic diseases that impair the function of methylmalonyl-CoA mutase, for which AdoCbl is coenzyme, produce methylmalonic aciduria (MMA). Impaired production of AdoCbl in MS does not cause MMA. There may have been some expectation that AdoCbl deficiency would cause MMA. However, in the CNS, the ATR enzyme in glial cells may not be as well protected from immune attack because of the plasma membrane being permeable or even the ATR enzyme being extracellular. There are a number of metabolic reactions known in bacteria with AdoCbl functioning as coenzyme. There might be similar reactions occurring in human glial cells, which have not yet been discovered and described, for example an amino transferase function. Autoimmune attack of ATR as the initial molecular lesion leading to the development of MS has never been proposed in the published literature. No scoping experiment or clinical trial of AdoCbl for therapy of MS patients has ever been reported in the published literature. The dramatic therapeutic effect of AdoCbl, when administered to a clinically definite MS patient, strongly indicates that there is much not understood regarding the activity of AdoCbl in the CNS.
Next, in the etiological sequence, an event occurs, which causes a breach of the BBB. Immune system cells from the systemic circulatory system infiltrate into the CSF. Some of the B lymphocytes, having immunologic memory, are on surveillance for ATR-like antigens. These are activated against self-antigens in the CSF. These immune system cells bind ATR enzyme in the CSF, undergo clonal expansion, release antibodies, and begin a cascade of events to attack ATR. The CSF becomes deficient in ATR and, subsequently, the CSF becomes deficient in AdoCbl. AdoCbl is necessary for the formation of normal healthy myelin.
The reaction of methylmalonyl-CoA mutase, which requires AdoCbl as a coenzyme is shown in Scheme 1.

A lack of AdoCbl means that methylmalonyl-CoA and propionyl-CoA accumulate. Propionyl-CoA could then replace succinyl-CoA and act as a primer for fatty acid synthesis. This would result in the synthesis of odd carbon number fatty acids. These odd carbon number fatty acids could be inserted into myelin sheaths producing abnormal myelin, which is antigenic. Hydrolysis of methylmalonyl-CoA produces elevated levels of methylmalonic acid, which inhibits normal fatty acid synthesis. There are other major changes in the myelin of MS patients, such as hypomethylation and citrullination (deimination of arginine), which may result from a cascade of events subsequent to the reduction of the AdoCbl concentration. Another possibility is the failure of an amino transferase anabolic reaction, which gives the appearance that citrullination has occurred. The abnormal myelin is antigenic and is subject to attack by the immune system, but is never completely destroyed. The normal regulatory events do not occur that stimulate production of T suppressor cells.
Poljakovic et al. (2006) reported that MS patients were 50% deficient in the concentration of growth hormone (GH) in the CSF. Zhang et al. (2006) reported that MS patients were deficient in the concentration of Interleukin-11. AdoCbl is a coenzyme for anabolic reactions in the CSF. It is probable that the reduced concentration of AdoCbl in the CSF is antagonistic to the normal synthesis of cytokines in the CSF, in a manner that is not fully understood. The strategy of therapy in this patent is to provide a normal or elevated concentration of AdoCbl and normal or elevated concentrations of growth hormone and other cytokines that are in reduced concentrations due to interference with their anabolic metabolism in the CSF. Cobalamin (Cbl) modulates synthesis of cytokines and growth factors. Cbl modulates in opposite ways the synthesis of the least Somme cytokines and growth factors. Therefore, these cytokines and growth factors can be defined as new cobalamin dependent CNS proteins, regardless of whether their synthesis increases or decreases in the presence of cobalamin.
Another AdoCbl reaction (called a MUT reaction) uses the adenosyl group from AdoCbl and methionine to produce S-adenosyl-methionine. The S-adenosyl-methionine is necessary for methylation of myelin sheath phospholipids. Failure of this reaction could result in the observed hypomethylation of myelin.
Failure of these reactions produces abnormal fatty acids, which can be incorporated into myelin. If the methylation of the myelin sheath phospholipids fails to occur, the resulting myelin will be fragile and antigenic. It is then subject to attack by the immune system causing demyelination. This may be referred to as conformational antigenicity.
There is a cascade of abnormal immune system events, stemming from the interaction of susceptibility genes, immune attack of ATR, deficiency of AdoCbl and some cytokines, an increase of necrosis factors, disturbance of anabolic metabolism thereby creating abnormal myelin, attack of abnormal myelin, etc. Ultimately, these events result in demyelination, oligodendrocyte death, axonal damage, and gliosis. The abnormal myelin likely has odd carbon number lipids, is hypomethylated, and has abnormal amination or deamination. Some B and T cells are activated against the abnormal myelin, because these immune cells, in the systemic blood system, may have been activated against similar viral or microbial antigens.
As the MS disease state progresses, an ascending peripheral neuropathy manifests with damage to axonal myelin and ultimately damage to denuded axons themselves. Loss of PNS myelin results in axonal death. In the CNS, the inflammation eventually becomes more diffuse and involves the whole brain with axonal injury and cortical demyelination.
Another fact demanding a logical explanation is the clinical similarity between subacute combined degeneration of the spinal cord and brain (SCD) and MS. SCD is a neurologic syndrome resulting from cobalamin (Vitamin B12) deficiency. Clinically, it is difficult to differentiate SCD from MS. Scheme 2 shows the pathways leading to the B12 coenzymes, AdoCbl and methylcobalamin (MeCbl).

When the concentration of Vitamin B12 is low, neither MeCbl nor AdoCbl are produced. The disease Pernicious Anemia results from failure to absorb Vitamin B12 and is often accompanied by megaloblastic anemia and neurological lesions. SCD is caused by Vitamin B12 deficiency and presents with neurological lesions and some anemia. SCD is treated by administration of Vitamin B12 and this rectifies the anemia and neurological lesions. In therapy of SCD with Vitamin B12 one clinical mistake is to also administer folic acid, which lessens anemia, but causes spinal cord lesions. The administration of folic acid decreases the AdoCbl concentration further by directing more Vitamin B12 along the pathway to produce MeCbl and directing less Vitamin B12 along the pathway to produce AdoCbl. This can be understood by consideration of Scheme 2. The fundamental and seminal CNS immune system event that leads to the development of MS is the autoimmune attack of ATR in the CSF. Consequently, Vitamin B12 is not converted to AdoCbl, no matter what concentration of Vitamin B12 is available.
Review of Neurological Inflammatory Diseases and Neurodegenerative Diseases
Neurological inflammatory diseases are widespread and include subacute combined degeneration of the spinal cord and brain (SCD) and multiple sclerosis (MS). SCD is a neurologic syndrome resulting from cobalamin (Vitamin B12) deficiency. The SCD neurologic syndrome involves multiple nerve pathways, includes sclerotic lesions in CNS white matter, and widespread uneven degeneration of myelin in the spinal cord and eventually in the brain. The degeneration of myelin and formation of sclerotic lesions is an inflammatory process, typical of a response to an infection. The pathological changes of SCD result in numbness, paraparesis (loss of motor functions in the legs), tingling “pins and needles,” clumsiness, unsteady gait, ataxia, and spasticity. Many of these neurologic abnormalities accompany pernicious anemia (PA), which results from extreme Vitamin B12 deficiency. MS presents with symptoms such as motor weakness, paraparesis, visual impairment, ataxia, and bladder dysfunction. The variety of sensory effects often includes numbness and tingling sensations. Fatigue is common. The numerous symptoms result from the various locations of demyelination and inflammation in the brain. MS symptoms may be similar to or identical to the symptoms of other neurological inflammatory diseases, such as SCD. Magnetic Resonance Imaging (MRI) provides imaging of the sclerotic lesions distributed in the white matter and spinal cord. The MRI of a MS patient may look very much like the MRI of persons with other neurological inflammatory diseases. The cerebrospinal fluid (CSF) of a MS patient contains much immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA); these immunoglobulins indicate immune system involvement in the disease.
Neurodegenerative diseases actually include the neurological inflammatory diseases as well as disease states of the brain, spinal cord and peripheral nervous system. Examples would be the brain dysfunction and degeneration in Alzheimer Disease (AD) and Amyotrophic Lateral Sclerosis (ALS), which affects the peripheral nervous system. There are many other neurodegenerative disorders of the brain, spinal cord and peripheral nervous system. In general, the nervous system must have an adequate supply of Vitamin B12 and more specifically its coenzymes MeCbl and AdoCbl. Several neurodegenerative disease states are known to result from deficiency in the concentration of Vitamin B12 and its coenzymes, and such deficiency might be a factor in many other neurodegenerative diseases.
AdoCbl may be involved directly or indirectly in the synthesis of myelin components, other growth factors, cytokines and regulatory peptides in the CSF. Pezacka et al. (1992) cultured glial cells in cobalamin deficient medium for 6 weeks and then subcultured them with a cobalamin-rich medium; data was said to suggest that glial cells were exquisitely sensitive to short-term cobalamin deprivation after which the two major coenzymes were not produced. Poljakovic (2006) showed that the concentration of Growth Hormone in the CSF of MS patients was 50% lower than in control subjects.
U.S. Pat. No. 6,908,611, Cruz, et al. teaches the use of Vitamin B12 compounds, including AdoCbl, in conjunction with “interferon compounds” to improve the effectiveness of the “interferon compounds” at a lower dose than normally administered. U.S. Pat. No. 6,908,611 does not teach that AdoCbl, by itself, provides effective therapy of MS and other neurological inflammatory diseases and neurodegenerative diseases, does not teach that AdoCbl facilitates remyelination, does not teach that AdoCbl stops secondary progression of disability in MS and allows extensive recovery from the symptoms of MS. U.S. Pat. No. 6,908,611 claims to demonstrate benefit in testing with the EAE animal model, but no human trials are provided, nor have human trials been reported in the literature.
U.S. Pat. No. 6,894,033, Cruz, et al. teaches the use of Vitamin B12 compounds, including AdoCbl, in conjunction with anti-inflammatory, anti-viral and anti-proliferative compounds to improve the effectiveness of the therapeutic compounds at a lower dose than normally administered. U.S. Pat. No. 6,894,033, does not teach that AdoCbl, by itself, provides effective therapy of MS and other inflammatory diseases, does not teach that AdoCbl facilitates remyelination, does not teach that AdoCbl stops secondary progression of disability in MS and allows extensive recovery from the symptoms of MS. U.S. Pat. No. 6,894,033, claims to demonstrate benefit in testing with the EAE animal model, but no human trials are provided, nor have been reported in the literature.
U.S. Pat. No. 7,368,531, Rosen and Rubin, teaches the use of human secreted polypeptides in conjunction with “therapeutics” (pharmaceutical compositions) for diagnosing and for treatment of diseases. Rosen and Rubin do not specify nor list the sequence for ATP: Cob (I) alamin adenosyltransferase (AdoCbl). Rosen and Rubin do list methionine adenosyltransferase, S-adenosylmethionine, the Interleukins IL-1, IL-1alpha, IL-1beta, IL-6, and IL-11. These compounds are not used in conjunction with AdoCbl. Rosen and Rubin do not teach that AdoCbl administered in combination with the Interleukins IL-1, IL-1alpha, IL-1beta, IL-6, and IL-11 is an effective therapeutic method for MS.
AdoCbl promotes and maintains myelination. Interleukins IL-1, IL-1alpha, IL-1beta, IL-6, and IL-11 also promote and maintain myelination. A deficiency in AdoCbl has the potential to cause a reduction in the production of Interleukins IL-1, IL-1 alpha, IL-1beta, IL-6, and IL-11. Therefore, the combination of AdoCbl and Interleukins IL-1, IL-1alpha, IL-1beta, IL-6, and IL-11 can be expected to cause remyelination and/or maintain myelination.
U.S. Pat. Nos. 7,199,098, 6,939,539, 6,620,847, 6,342,476, 6,362,161, 6,054,430, 5,981,589, and 5,800,808 all by Konfino et al. teach the modification and use of Copolymer-1 (a synthetic polypeptide analog of myelin basic protein (MBP), which is a natural component of the myelin sheath). These are based on U.S. Pat. No. 3,849,550 by Teitelbaum, et al.
These patents claim to demonstrate benefit in testing with the EAE animal model, and human trials have been reported in the literature. Copolymer-1's use for therapy of MS has been approved by the Food and Drug Administration (FDA). These patents do not teach that AdoCbl facilitates remyelination, do not teach that AdoCbl stops secondary progression of disability in MS and that AdoCbl allows extensive recovery from the symptoms of MS. Human trials have been reported in the literature demonstrating that Copolymer-1 is effective in lowering the rate of relapse in Relapsing Remitting Multiple Sclerosis (RRMS), but shows no effective benefit for the Chronic Progressive forms of MS.
Copolymer-1 (glatiramer acetate) is the acetate salts of synthetic polypeptides from four amino acids: (Glu-Ala-Lys-Tyr)x-CH3COOH and is analogous to MBP. Schubert and Hill (2006) indicate that four residues appear to bind to AdoCbl in the ATR enzyme. Several of the point mutations causing Methyl Malonic Aciduria (MMA) are in the 186 to 194 residue region. The binding sites include Gly 97, Ser 174, Arg 186, Arg 190, Arg 191, Gln 193, and Gln 234. Two regions of interest in this current patent are highly conserved; first is a sequence of five amino acids (Sequence 4 in the Sequence Listing) in the 190 to 194 amino acids RRAER=Arg-Arg-Ala-Glu-Arg and the second region includes additional amino acids totaling nine amino acids (Sequence 3 in the Sequence Listing) in the 186 to 194 region RAVCRRAER=Arg-Ala-Val-Cys-Arg-Arg-Ala-Glu-Arg. The concept for two claims is to use these amino acids or these sequences to form synthetic polypeptides like Copolymer-1, referred to as Copolymer-3-ATR (3 types of amino acids) and referred to as Copolymer-5-ATR (5 types of amino acids). These sequences form a cleft on human ATR that binds AdoCbl. The rationale is that this sequence is invariant between all microbial, mouse, and human PduO class ATRs. Therefore, an antibody to this sequence should be cross reactive to human ATR. The mechanism of action by Copolymer-1 has been opined as causing the formation of protective antibodies. Any of Sequences 1 through 6 in the Sequence Listing could be used. The effectiveness of Copolymer-1 was first demonstrated on the EAE animal model. EAE may not be a suitable animal model to demonstrate effectiveness of these epitopes. The totally gastectomized (TGX) rat is possibly a suitable animal model to demonstrate effectiveness of these epitopes, despite the lack of autoimmune attack of ATR. An animal model of MS could be created by inoculation of a rat, or other animal with microbial ATR-like enzymes (Sequences 7 through 14 in the Sequence Listing), followed by injecting its blood into its cranial CSF and thereby inducing autoimmune attack of ATR. This MS animal model would be suitable to evaluate Copolymer-3-ATR and Copolymer-5-ATR. This MS animal model would be suitable to evaluate the effectiveness of AdoCbl therapy.
The compound defined for one claim is Copolymer-3-ATR, which is the acetate salts of synthetic polypeptides from three amino acids: arginine, alanine, and y-benzyl glutamate in a molar ratio of approximately 3:1:1, respectively. The second compound defined for a claim is Copolymer-5-ATR, which is the acetate salts of synthetic polypeptides from five amino acids: arginine, alanine, valine, S-acetamidomethyl cysteine, and y-benzyl glutamate in a molar ratio of approximately 4:2:1:1:1, respectively. It is synthesized by chemically polymerizing the five amino acids forming products with average molecular weights of 23,000 daltons (U.S. Pat. No. 3,849,550). The designed structure for Copolymer-3-ATR is (Arg-Arg-Ala-Glu-Arg)x-CH3COOH and is analogous to ATR. Copolymer-3-ATR may be prepared by methods known in the art, for example, the process disclosed in U.S. Pat. No. 3,849,550, wherein the N-carboxyanhydrides of alanine, y-benzyl glutamate and E-N-continued trifluoro-acetylarginine are polymerized at ambient temperature in anhydrous dioxane with diethylamine as initiator. The deblocking of the y-carboxyl group of the glutamic acid is effected by hydrogen bromide in glacial acetic acid and is followed by the removal of the trifluoroacetyl groups from the arginine residues by 1M piperidine. For the purposes of this application, the terms “ambient temperature” and “room temperature” should be understood to mean a temperature ranging from about 20 to 26 degree C.
The designed structure for Copolymer-5-ATR is (Arg-Ala-Val-Cys-Arg-Arg-Ala-Glu-Arg)x-CH3COOH and is analogous to ATR. Copolymer-5-ATR may be prepared by methods known in the art, for example, the process disclosed in U.S. Pat. No. 3,849,550, wherein the N-carboxyanhydrides of alanine and valine, S-acetamidomethyl cysteine, y-benzyl glutamate and E-N-trifluoro-acetylarginine are polymerized at ambient temperature in anhydrous dioxane with diethylamine as initiator. The deblocking of the y-carboxyl group of the glutamic acid is effected by hydrogen bromide in glacial acetic acid and is followed by the removal of the trifluoroacetyl groups from the arginine residues by 1M piperidine. The deblocking of the S-acetamidomethyl group from cysteine is accomplished with iodine. For the purposes of this application, the terms “ambient temperature” and “room temperature” should be understood to mean a temperature ranging from about 20 to 26 degree C.
The invention will be exemplified but not necessarily limited by the following examples.